The glycoprotein erythropoietin (“EPO”) has a molecular mass of about 34,000 Daltons and is composed of 165 amino acids. It acts as the primary hormone in the regulation of erythropoiesis, in which cell differentiation and proliferation results in increased red blood cell production. EPO is produced by the kidneys during times of low oxygen levels in the blood. EPO binds to the EPO receptor (“EPOR”), reportedly inducing dimerization of two receptors that initiate a cascade leading to the synthesis of hemoglobin and increased production of mature erythrocytes, and consequently higher oxygen levels in the blood. See, e.g., Graber and Krantz, Ann. Rev. Med. 29, 51-66(1978).
Recombinant human EPO, sometimes referred to as rhEPO and sold under the brand names EPOGEN® (epoetin alfa) and PROCRIT® (epoetin alfa), is widely used in the treatment of patients suffering from anemia, such as anemia from impaired renal function, cancer chemotherapy, or AZT treatment. These drugs are administered either intravenously or subcutaneously.
Efforts have been made to identify alternative peptide EPOR agonists or binding molecules that would improve upon the activity or characteristics of EPO. Erythropoietin (EPO) and a description of novel peptides that bind to the EPO receptor (EPOR) have been described in U.S. Pat. Nos. 7,084,245; 7,414,105; and 7,459,522, each of which are incorporated herein by reference. An X-ray crystal structure of a peptide agonist complexed with the extracellular domain of the EPO receptor has also been published. See Livnah et al., Science, 273, 1996, 464-471; the crystal structure coordinates are hereby incorporated by reference.
The structure shows a homodimeric complex containing two ligands and two EPOR domains with near perfect C2 symmetry. A hydrogen bond exists between the two Gln18 residue sidechains of the dimer near the C-terminal regions of the ligands. The C-terminal regions project away from the binding site towards solution. Based on the near perfect C2 symmetry of the homodimer complex, it is possible that dimeric EPO analogues with pseudo C2/C2 symmetry may be favored due to an enhanced entropic component to binding.
Wrighton et al. describe a series of small peptides that might act as mimics of EPO. Science, 273, 458 (1996). The compounds were identified using a phage display library and are represented by a family of 16 amino acid peptides having an intramolecular disulfide bond located between two cysteine residues with eight residues between them. U.S. Pat. No. 7,084,245 describes a group of three peptide dimers that are agonists of EPOR. The peptides each contain intramolecular disulfide linkages, and the peptides were covalently joined by a linker at their carboxy terminus. The phage display technology used to identify these EPOR agonists was based on a NNK codon library that was biased towards certain amino acid residues. Moreover, the number of molecules required for a perfect random library of peptides of 14 residues exceeds the numerical limitations of the phage. In addition, 60% of mRNA nucleotide sequences are estimated to include at least one stop codon in a standard NNK library. Thus, it is clear that the number of known EPOR binding compounds, including EPOR agonists, is limited and new molecules are needed.
Moreover, even the compounds that have been identified have limited stability, thereby making their production and handling more difficult and limiting the ways in which they can be administered and used. For instance, the known EPOR agonists contain peptide sequences having intramolecular disulfide linkages which are thought to be necessary for binding. Unfortunately, these disulfide bonds are unstable and can undergo cleavage in a facile manner depending on the conditions, such as under reducing conditions. In addition, peptides as a class are susceptible to acid hydrolysis and enzymatic degradation by proteases which limits the half-life of the drug in the body and further limits the ways they can be administered. Consequently, new EPOR binding compounds, including EPOR agonists, are needed with increased stability, especially molecules that have more stable intramolecular and intermolecular bonds.
It is also known in the art that a patient's hematocrit (i.e., the amount of red blood cells in whole blood, measured as a percentage of the whole blood) and the change in hematocrit can be monitored using readily available, non-invasive finger cuffs that test for hemoglobin levels (a surrogate for hematocrit. These cuffs are also known as pulse oximeters, and commercially available versions are available both over the counter and as a prescription medical device.